High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing

Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

Paternal chromosome elimination and X non-disjunction on asymmetric spindles in Sciara male meiosis

Meiosis in male Sciara is unique with a single centrosome. A monopolar spindle forms in meiosis I, but a bipolar spindle forms in meiosis II. The imprinted paternal chromosomes are eliminated in meiosis I; there is non-disjunction of the X in meiosis II. Despite differences in spindle construction and chromosome behavior, both meiotic divisions are asymmetric, producing a cell and a small bud. Observations of live spermatocytes made with the LC-PolScope, differential interference contrast optics and fluorescence revealed maternal and paternal chromosome sets on the monopolar spindle in meiosis I and formation of an asymmetric monastral bipolar spindle in meiosis II where all chromosomes except the X congress to the metaphase plate. The X remains near the centrosome after meiosis I and stays with it as the spindle forms in meiosis II. Electron microscopy revealed amorphous material between the X and the centrosome. Immunofluorescence with an antibody against the checkpoint protein Mad2 stains the centromeres of the maternal X dyad in late meiosis I and in meiosis II where it fails to congress to the metaphase plate. Mad2 is also present throughout the paternal chromosomes destined for elimination in meiosis I, suggesting a possible role in chromosome imprinting. If Mad2 on the X dyad mediates a spindle checkpoint in meiosis II, it may delay metaphase to facilitate formation of the second half spindle through a non-centrosomal mechanism.

A case report on Wolf-Hirschhorn syndrome

Wolf-Hirschhorn syndrome is a genetic condition that affects many systems of the human body. It is caused by a deletion of the band 4p16.3 and this deletion may be sub microscopic. Individuals affected by the syndrome have a special phenotype: wide bridge of the nose, widely spaced eyes, micrognathia, microcephaly, growth retardation, cryptorchidism, heart defects, hearing loss and severe intellectual disability. A familial translocation is seen in 5-13% of the patients. Other patients have de novo deletions, usually on the paternal chromosome 4, or de novo translocations in 1.6%. Prenatal diagnosis is possible. We are hereby reporting a case of 9 months old infant who showed delayed physical and neurocognitive development and a characteristic appearance, which led to the diagnosis of this genetic disease.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato, reduction to diploidy (dihaploidy) allows hybridization to diploid germplasm, introgression breeding, and may facilitate the production of inbreds. Pollination with haploid inducers yields maternal dihaploids, as well as triploid and tetraploid hybrids. It is not known if dihaploids result from parthenogenesis, entailing development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of haploid inducer DNA in some of the dihaploids. We characterized the genomes of 1,001 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three haploid inducers, IVP35, IVP101, and PL4. We detected inheritance of full or partial chromosomes from the haploid inducer parent in 0.87% of the overall dihaploid progeny, irrespective of the combination of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny. Residual haploid inducer DNA is consistent with genome elimination as the mechanism of haploid induction. Further, the fact that paternal chromosome breaks are specific to dihaploids and tetraploid progeny suggests that they may be specific to 2x sperms, and supports the hypothesis that 2x sperms facilitate genome elimination.

Single-molecule sequencing of long DNA molecules allows high contiguity de novo genome assembly for the fungus fly, Sciara coprophila

ABSTRACTThe lower Dipteran fungus fly, Sciara coprophila, has many unique biological features. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in aiding these studies. We approached assembling the Sciara genome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of the Sciara genome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in the Sciara genome since DNA modifications may play a role in imprinting in Sciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

CTCF controls imprinted gene activity at the mouse Dlk1-Dio3 and Igf2-H19 domains by modulating allele-specific sub-TAD structure

SUMMARYMammalian genomic imprinting is essential for development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. Here, we compared chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints—the Igf2-H19 and the Dlk1-Dio3 domains—and assessed the involvement of the insulator protein CTCF. At both domains, CTCF binds the maternal allele of a differentially-methylated region (DMR), in addition to multiple instances of bi-allelic CTCF binding in their surrounding TAD (Topologically Associating Domain). On the paternal chromosome, bi-allelic CTCF binding alone is sufficient to structure a first level of sub-TAD organization. Maternal-specific CTCF binding at the DMRs adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal sub-TAD organisation. Genome-editing experiments at the Dlk1-Dio3 locus confirm that the maternal sub-TADs are essential during development to maintain the imprinted Dlk1 gene in an inactive state on the maternal chromosome.